Structure Determination of the Human Protective Protein: Twofold Averaging Reveals the Three-Dimensional Structure of a Domain Which was Entirely Absent in the Initial Model

Mutations in the human `protective protein' result in the human lysosomal storage disease galactosialidosis. The structure of the human `protective protein' has been determined using X-ray crystallography to a resolution of 2.2 Å. Initial phases were obtained from molecular replacement calculations. A very partial search model comprising 30% of the scattering mass, was constructed from the atomic model of the wheat serine carboxypeptidase. This truncated probe was used to find the position of two monomers in the asymmetric unit. Subsequently, `bootstrapping' cycles, consisting of twofold averaging and model expansion, retrieved the electron density for residues initially missing. In particular, it proved possible to add a domain (more than 110 residues) to the initial partial search model. In total, 314 residues per asymmetric unit were added to the 588 residues of the initial model. Factors contributing to our success are discussed.